Team:Cambridge/Protocols

From 2009.igem.org

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(Carotene extraction using acetone)
(Carotene extraction using acetone)
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*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums
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== Carotene extraction using acetone ==
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== Carotene extraction with acetone ==
Adopted from ''Yuan et al. (2006)'':
Adopted from ''Yuan et al. (2006)'':

Revision as of 22:31, 3 August 2009


Protocols

Producing competent cells

Starting from a single colony on a plate:

  • Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight
  • Take 10ml of the culture and innoculate into larger LB volume and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)
  • Put culture on ice for 30 minutes
  • Centrifuge at 4000g for 6 minutes
  • Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES
  • Repeat centrifugation
  • Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES
  • Repeat centrifugation
  • Resuspend cells in ice-cold 10% glycerol (20ml)
  • Combine to form two tubes of 40ml glycerol
  • Repeat centrifugation
  • Resuspend in ice-cold glycerol (3ml)
  • Divide cells into 100ul aliquots and store at -80

(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)

Competent cells Transformation

  • Electrocompetent cells thawed on ice
  • Prepare vector DNA on ice
  • Biobricks
  • With pipette tip, punch hole through foil cover into designated well
  • Add 20uL DIW
  • We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C
  • Violacein and melanin need to be thawed
  • Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total
  • 5uL of biobricks
  • 0.5uL of melanin and violacein plasmid
  • Add 45 uL Competent cells
  • Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles
  • Thoroughly dry the cuvette
  • 1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF
  • Add 0.25 mL SOC liquid medium to electrocuvette
  • Incubate electrocuvettes at 37 degrees C for 60 minutes
  • Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader
  • Orange genes biobrick: ampicillin
  • Promoter for orange genes biobrick: ampicillin
  • Melanin: ampicillin, copper, and tyrosine
  • Violacin: trimethoprim
  • Do 1:10 dilution with SDW into a new eppendorf
  • Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums

Carotene extraction with acetone

Adopted from Yuan et al. (2006):

1. Incubate E.coli in 5ml LB with antibiotics at 37 oC for 20 hours.

2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.

3. Re-suspend cells in 300 ul acetone and vortex for 5 minutes*** (***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products), but we did not have the equipment. Vortex is used for homogenisation instead).

4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.

5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using Omega Microplate Readers from BMG-Labtech.

CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. Acetone is also highly volatile. We are in the process of resolving these issues.